Reversibly inactivated acidified plasmin

ABSTRACT

The present invention provides a fibrinolytic composition useful as a therapeutic for administration to a patient having a thrombotic occlusion. In one aspect of the present invention, the fibrinolytic composition comprises a reversibly inactivated acidified serine protease substantially free of a plasminogen activator, a low buffering capacity buffer, and optionally, a stabilizing agent. In another aspect of the invention, the fibrinolytic composition of the present invention comprises a reversibly inactivated acidified plasmin substantially free of a plasminogen activator, a low buffering capacity buffer, and optionally, a stabilizing agent.

REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of International ApplicationPCT/US00/31090, filed Nov. 13, 2000, and published in English on May 25,2001, which in turn is a continuation-in-part of U.S. patent applicationSer. No. 09/438,331 filed Nov. 13, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates generally to compositions useful inthrombolytic therapy. More particularly, the present invention isdirected to novel compositions comprising reversibly inactivatedacidified serine protease compositions useful in clot dissolutiontherapy wherever directed delivery to a thrombus is feasible.

BACKGROUND

[0003] The blood clotting process, as a mechanism of hemostasis or inthe generation of a pathological condition involving thrombi, requirestwo cooperating pathways: 1) following activation, triggered by theenzyme thrombin, circulating platelets adhere to one another accompaniedby the release of factors like thromboxane A2 and the subsequentformation of a plug created from the aggregated platelets, and 2) theactivation of a cascade of proteolytic enzymes and cofactors, most ofwhich are plasma glycoproteins synthesized in the liver, to produce athrombus. Thrombi are composed mainly of an insoluble fibrin network,which entraps circulating blood cells, platelets, and plasma proteins toform a thrombus.

[0004] Thromboembolic disease, i.e., the pathological blockage of ablood vessel by a blood clot, is a significant cause of mortality andmorbidity. Most spontaneously developing vascular obstructions are dueto the formation of intravascular blood clots, or thrombi. Smallfragments of a clot may also detach from the body of a clot and travelthrough the circulatory system to lodge in distant organs and initiatefurther clot formation. Myocardial infarction, occlusive stroke, deepvenous thrombosis (DVT) and peripheral arterial disease are well-knownconsequences of thromboembolic phenomena.

[0005] Plasminogen activators are currently the favored agents employedin thrombolytic therapy, all of which convert plasminogen to plasmin andpromote fibrinolysis by disrupting the fibrin matrix (Creager M. A. &Dzau V. J., Vascular Diseases of the Extremities, ppgs. 1398-1406 inHarrison's Principles of Internal Medicine, 14^(th) ed., Fauci et al,editors, McGraw-Hill Co., New York, 1998; the contents of which isincorporated herein by reference in its entirety). The most widely usedplasminogen activators include a recombinant form of tissue-typeplasminogen activator (tPA), urokinase (UK) and streptokinase (SK), aswell as a new generation of plasminogen activators selected for improvedpharmacokinetics and fibrin-binding properties. All of these plasminogenactivators, however, act indirectly to effect lysis and require anadequate supply of their common substrate, plasminogen, at the site ofthe thrombus.

[0006] UK and tPA convert plasminogen to plasmin by cleaving theArg⁵⁶¹-Val⁵⁶² peptide bond. The resulting two polypeptide chains ofplasmin remain joined by two interchain disulfide bridges. The lightchain of 25 kDa carries the catalytic center and is homologous totrypsin and other serine proteases. The heavy chain (60 kDa) consists offive triple-loop kringle structures with highly similar amino acidsequences. Some of these kringles contain so-called lysine-binding sitesthat are responsible for plasminogen and plasmin interaction withfibrin, α₂-antiplasmin or other proteins. Variant forms of truncatedplasmin, including variants lacking some or all of the kringle regionsof the plasmin heavy chain, are disclosed by Wu et al. in U.S. Pat. No.4,774,087, incorporated herein by reference in its entirety. SK andstaphylokinase activate plasminogen indirectly by forming a complex withplasminogen, which subsequently behaves as a plasminogen activator toactivate other plasminogen molecules by cleaving the arginyl-valinebond.

[0007] Plasmin is a different mechanistic class of thrombolytic agentthat does not activate plasminogen. Plasmin directly cleaves fibrin in athrombus, resulting in lysis. This avoids the requirement forplasminogen or plasminogen activators to be present in a thrombus. Manyclots that are deficient in plasminogen due to thrombus contractiontriggered by platelets and by Factor VIII.

[0008] Although tPA, SK and UK have been successfully employedclinically to reduce a thrombotic occlusion, serious limitations persistwith their use in current thrombolytic therapy. For example, because thesystemic administration of tPA is not specifically targeted to thethrombus, it can result in significant systemic hemorrhage. Otherlimitations associated with plasminogen activators impact their overallusefulness. At best, the use of current thrombolytic therapy results inrestored vascular blood flow within 90 minutes in only about 50% ofpatients, while acute coronary re-occlusion occurs in roughly 10% of thepatients. Coronary recannulization requires on average 45 minutes ormore, and intracerebral hemorrhage occurs in 0.3% to 0.7% of patients.Residual mortality is still about 50% of the mortality level in theabsence of thrombolysis treatment.

[0009] A different approach that avoids many of the problems associatedwith the systemic administration of a plasminogen activator is togenerate plasmin at the site of the thrombus or to directly administerthe plasmin either into or proximally to the thrombus. Reich et al. inU.S. Pat. No. 5,288,489 discloses a fibrinolytic treatment that includesparenteral administration of plasmin into the body of a patient. Theconcentration and time of treatment were sufficient to allow activeplasmin to attain a concentration at the site of an intravascularthrombus that is sufficient to lyse the thrombus or to reducecirculating fibrinogen levels. Reich et al. require generation of theplasmin from plasminogen immediately prior to its introduction into thebody.

[0010] In contrast, Jenson in U.S. Pat. No. 3,950,513 discloses aporcine plasmin preparation that is asserted to be stabilized at low pH.However, such plasmin solution must be neutralized before systemicadministration to humans for thrombolytic therapy.

[0011] Yago et al. in U.S. Pat. No. 5,879,923 discloses plasmincompositions employed as a diagnostic reagent. The compositions of Yagoet al. consist low concentrations of plasmin at a neutral pH and anadditional component that may be 1) an oligopeptide consisting of atleast two amino acids, or 2) at least two amino acids, or 3) a singleamino acid and a polyhydric alcohol, and the amino acids arespecifically identified.

[0012] Numerous technical problems, such as the difficulty of preparingplasmin free of contaminating plasminogen activators, have preventedclinical use of plasmin . Plasmin preparations were typicallyextensively contaminated by the plasminogen activators streptokinase andurokinase, resulting in the attribution of thrombolytic activity to thecontaminating plasminogen activators rather than to plasmin itself. Thecontaminating plasminogen activators can also trigger systemic bleedingat sites other than the targeted thrombosis. One factor limitingclinical use of plasmin is that plasmin, as a serine protease with broadspecificity, is highly prone to autodegradation and loss of activity atphysiological pH when prepared as a highly purified and highlyconcentrated solution. This provides severe challenges to the productionof high-quality plasmin, to the stable formulation of this activeprotease for prolonged periods of storage prior to use, and to safe andlocalized administration of plasmin to human patients suffering fromocclusive thrombi.

[0013] Thus, there is a need for a therapeutic composition comprising astabilized serine protease capable of cleaving fibrin and apharmaceutically acceptable carrier with a pH range sufficiently low toreversibly inactivate the serine protease, yet sufficiently high tolimit acid hydrolysis of peptide bonds within the serine protease.Further, there is a need for such therapeutic composition to have a lowbuffer capacity to maintain low pH during storage, yet permit plasmin torapidly revert to its active form at the pH in the local environment ofthe clot.

[0014] There is also a need for a therapeutic composition comprising areversibly inactivated acidified serine protease stabilized by at leastone pharmaceutically acceptable stabilizing agent and a pharmaceuticallyacceptable carrier.

[0015] These and other objectives and advantages of the invention willbecome fully apparent from the description and claims that follow or maybe learned by the practice of the invention.

SUMMARY OF THE INVENTION

[0016] This invention overcomes the disadvantages of the prior art byproviding a fibrinolytic composition which can be therapeuticallyadministered directly at or proximal to a site of a thromboticocclusion. Further, the fibrinolytic composition of the presentinvention has a substantially long-term shelf life with respect to theprior art.

[0017] In one aspect of the present invention, the fibrinolyticcomposition comprises a reversibly inactivated acidified serine proteasesubstantially free of a plasminogen activator, a low buffering capacitybuffer, and optionally, a stabilizing agent. Such serine proteasesinclude trypsin, chymotrypsin, pancreatic elastase II, cathepsin G,prostate-specific antigen, leukocyte elastase, chymase, tryptase,acrosin, human tissue kallikrein, and plasmin. Plasmin includesGlu-plasmin or Lys-plasmin, derivatives and modified or truncatedvariants thereof, including, but not limited to, midi-plasmin,mini-plasmin, or micro-plasmin.

[0018] In another aspect of the invention, the fibrinolytic compositionof the present invention comprises a reversibly inactivated acidifiedplasmin substantially free of a plasminogen activator, a low bufferingcapacity buffer, and optionally, a stabilizing agent. Plasmin includesGlu-plasmin or Lys-plasmin, derivatives and modified or truncatedvariants thereof, including, but not limited to, midi-plasmin,mini-plasmin, or micro-plasmin.

[0019] Buffers employed in the present invention include such lowbuffering capacity buffers which are present in the composition at aconcentration at which the pH of the composition is rapidly raised to aneutral pH by adding no more than about an equal volume of serum to thecomposition. In one aspect of the invention, the buffer comprises atleast one pharmaceutically acceptable acid, such as an amino acid, aderivative of the at least one amino acid, a dipeptide, an oligopeptidewhich includes the at least one amino acid, and combinations thereof.Amino acids employable as the buffer include serine, threonine,methionine, glutamine, glycine, isoleucine, valine, aspartate, andalanine. Other low buffering capacity acids may be employed and includeformic acid, acetic acid, citric acid, hydrochloric acid, lactic acid,malic acid, tartaric acid, benzoic acid, derivatives thereof, andcombinations thereof. The amino acids and the other low bufferingcapacity acids may be combined in any desired combination as well.

[0020] Stabilizing agents which may be employed in the present inventioninclude pharmaceutically acceptable carbohydrates, salts, glucosamine,thiamine, niacinamide, citrulline, and combinations thereof.

[0021] Thus, a unique fibrinolytic composition is now provided thatsuccessfully addresses the shortcomings of existing compositions andprovides distinct advantages over such compositions. Additional objects,features, and advantages of the invention will become more apparent uponreview of the detailed description set forth below when taken inconjunction with the accompanying drawing figures, which are brieflydescribed as follows.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 illustrates the pH dependence of plasmin activity asmeasured with the chromogenic substrate S2251.

[0023]FIG. 2 illustrates plasmin stability in acidified saline (pH 3.7)as measured by a caseinolytic assay.

[0024]FIG. 3 illustrates that pH stability of plasmin does not depend onthe buffering agent nor concentration of buffering agent.

[0025]FIG. 4 illustrates the effectiveness of plasmin or tPA plusplasminogen in thrombolysis.

[0026]FIG. 5 illustrates the stability at 37° C. of a reversiblyinactivated acidified plasmin at pH of 3.7, with carbohydratestabilizers.

[0027]FIG. 6 illustrates the stability at 37° C. of a reversiblyinactivated acidified plasmin at a pH of 3.7 with glucosamine,niacinamide, thiamine or citrulline as a stabilizing agent.

[0028]FIG. 7 illustrates the progressive degradation of a plasmincomposition at a pH of 2.2, 3.5, or 3.7.

[0029]FIG. 8 illustrates the cleavage sites generated in plasmin at pH2.2 and 3.8.

[0030]FIG. 9 illustrates the titration, with human serum, of plasminsolutions having various low buffering capacity buffers.

[0031]FIG. 10 compares the thrombolytic potency of plasmin in saline atpH 3.7 with plasmin neutralized before injection into the clot.

DETAILED DESCRIPTION

[0032] A full and enabling disclosure of the present invention,including the best mode known to the inventors of carrying out theinvention is set forth more particularly in the remainder of thespecification, including reference to the Examples. This description ismade for the purpose of illustrating the general principles of theinvention and should not be taken in the limiting sense.

[0033] The present invention addresses the need for a fibrinolyticcomposition that is stable on storage and can be therapeuticallyadministered to a patient having a thrombotic occlusion. Therefore, inone aspect the present invention provides a fibrinolytic compositioncomprising a reversibly inactivated acidified serine proteasesubstantially free of a plasminogen activator, a low buffering capacitybuffer, and optionally, a stabilizing agent. Such serine proteasesinclude trypsin, chymotrypsin, pancreatic elastase II, cathepsin G,prostate-specific antigen, leukocyte elastase, chymase, tryptase,acrosin, human tissue kallikrein, and plasmin. Plasmin includesGlu-plasmin or Lys-plasmin, derivatives and modified or truncatedvariants thereof, including, but not limited to, midi-plasmin,mini-plasmin, or micro-plasmin.

[0034] The present invention further provides a fibrinolytic compositioncomprising a reversibly inactivated acidified serine proteasesubstantially free of plasminogen activator and a pharmaceuticallyacceptable acidified carrier, further comprising a pharmaceuticallyacceptable stabilizing agent.

[0035] In another aspect of the present invention invention, thefibrinolytic composition of the present invention comprises a reversiblyinactivated acidified plasmin substantially free of a plasminogenactivator, a low buffering capacity buffer, and optionally, astabilizing agent. Again, plasmin includes Glu-plasmin or Lys-plasmin,derivatives and modified or truncated variants thereof, including, butnot limited to, midi-plasmin, mini-plasmin, or micro-plasmin.

[0036] Buffers employed in the present invention include such lowbuffering capacity buffers which are present in the composition at aconcentration which would allow the pH of the composition to be changedto a physiological pH by contacting body fluids. In an aspect of theinvention, the buffer comprises at least one pharmaceutically acceptableacid, such as an amino acid, a derivative of the at least one aminoacid, a dipeptide, an oligopeptide which includes the at least one aminoacid, and combinations thereof. Amino acids employable as the bufferinclude serine, threonine, methionine, glutamine, alanine, glycine,isoleucine, valine, aspartate, alanine, and combinations thereof. Otherlow buffering capacity acids may be employed and include formic acid,acetic acid, citric acid, hydrochloric acid, lactic acid, malic acid,tartaric acid, benzoic acid, derivatives thereof, and combinationsthereof. The amino acids and the other low buffering capacity acids maybe combined in any desired combination as well.

[0037] Stabilizing agents which may be employed in the present inventioninclude pharmaceutically acceptable carbohydrates, salts, glucosamine,thiamine, niacinamide, citrulline, and combinations thereof. Furtherstabilizing agents include, but are not limited to, monosacchrides,disacchrides, polysacchrides, polyhydric alcohols, or combinationsthereof. For example, such stabilizing agents include sugars or sugaralcohols, such as glucose, maltose, mannitol, sorbitol, sucrose,lactose, trehalose, or combinations thereof. Salts, such as sodiumchloride, potassium chloride, magnesium chloride, calcium chloride, orcombinations thereof, are employable as stabilizing agents in thepresent invention.

[0038] With the escalating use of arterial and venous catheters in theclinics, local delivery of an active plasmin in close proximity to, oractually into, a thrombus offers an attractive therapeutic opportunityin thrombolytic therapy. Being an active serine protease, plasmin is adirect thrombus-dissolving agent, in contrast to plasminogen activatorsthat require the presence of the zymogen plasminogen in the vicinity ofthe thrombus. Local catheter-directed thrombolytic therapy with activeplasmin can be regulated to achieve total thrombolysis, and plasmin hasthe potential to be a safer thrombolytic agent because the lower dosagerequired for local delivery may significantly reduce bleedingcomplications frequently associated with high dose thrombolytic therapyinduced by plasminogen activators. Furthermore, any potential spillageof plasmin from the immediate vicinity of the thrombus site will bequickly neutralized by circulating α₂-antiplasmin.

[0039] In the past, there have been several technical challengesassociated with plasmin purification, and storage, as well as with itstherapeutic use and delivery. Plasmin is an active serine protease andis subject to autodigestion and inactivation at a physiological pH.Plasmin degradation, unfortunately, is also most evident in the pH rangerequired for in vivo thrombolysis.

[0040] The fibrinolytic composition, as incorporated into the presentinvention, includes the maintenance of the plasmin in an acidic bufferduring purification, as well as its formulation in an acidified carrierhaving a pharmaceutically acceptable low buffering capacity buffer,thereby providing a reversibly inactivated acidified plasmin-containingfibrinolytic composition substantially free of plasminogen activator. Itis contemplated to be within the scope of the present invention for thefibrinolytic composition to be a lyophilized composition that may bereconstituted by the addition of a pharmaceutically acceptable carriessuch as, but not limited to, water, physiological saline or any othersolvent that will allow administration of the composition to a human oranimal. Its efficacy in restoring vascular patency was demonstrated inin vitro assays and in an in vivo rabbit jugular vein thrombolysismodel.

[0041] The term “reversibly inactivated” as used herein refers to anenzymatic activity that is substantially free of activity under aspecific set of conditions but will revert to an active form whentransferred to another set of conditions.

[0042] The term “pharmaceutically acceptable carrier” as used hereinrefers to any carrier that is physiologically tolerated by a recipienthuman or animal, including, but not limited to, water, salt solutions,physiological saline, or any other liquid or gel in which a fibrinolyticagent such as plasmin may be dissolved or suspended. The“pharmaceutically acceptable carrier” may include any pharmaceuticallyacceptable compound that will give a plasmin solution having a pH belowabout 4.0 and which has low or zero buffering capacity.

[0043] The term “physiological pH” as used herein refers to a pH betweenabout pH 6.5 and about 7.5, ore typically between about pH 7.1 and about7.5.

[0044] The term “body fluid” as used herein refers to any body fluidincluding, but not limited to, blood, serum, plasma, semen and urine.

[0045] The term “low pH buffering capacity buffer” or “low bufferingcapacity buffer” as used herein refers to the amount of acid or basethat a buffer can neutralize before the pH begins to change to anappreciable degree. As used herein a low buffering capacity buffer willbe significantly pH adjusted by the addition of a small volume of anacid or base relative to the volume of the low buffering capacity buffersolution. For example, in the present invention, the buffer is presentin the composition at a concentration that would allow the pH of thecomposition to be changed by contacting a body fluid. This term is meantto include solutions acidified by strong acids including, but notlimited to, hydrochloric acid, nitric acid and sulfuric acid, and whichhave no buffering capacity.

[0046] The term “thrombus” as used herein refers to a thrombus in ablood vessel or device contacting blood (e.g. catheter devices orshunts). A thrombus may comprise fibrin and may further comprise, but isnot limited to, platelets, erythrocytes, lymphocytes, lipid or anycombination thereof. A “thrombus” may be, but is not limited to, anannular thrombus, ball thrombus, hyaline thrombus, mural thrombus,stratified thrombus or white thrombus.

[0047] The term “thrombotic occlusion” as used herein refers to apartial or total blockage of a vessel due to the formation of athrombotic clot, wherein the thrombus comprises at least fibrin. Thevascular vessel occluded may be, but is not limited to, a vein, artery,venule, arteriole, capillary, vascular bed or the heart and may bewithin any vascularized organ or tissue of the human or animal body. Thethrombotic occlusion may also be of a catheter or other implantincluding, but not limited to, prosthetic vessels and grafts ofsynthetic, human or animal origin and effectively blocked by anocclusion comprising fibrin.

[0048] The term “catheter device” as used herein refers to any catheteror tube-like device that may enter the body, and includes but is notlimited to, an arterial catheter, cardiac catheter, central catheter,central venous catheter, intravenous catheter, peripherally insertedcentral catheter, pulmonary artery catheter or tunneled central venouscatheter and arterio-venal shunts.

[0049] The term “pharmaceutically acceptable acidified carrier” as usedherein refers to any pharmaceutically acceptable carrier that has beenacidified to a pH below about 4.0. The “pharmaceutically acceptableacidified carrier” may comprise a low or zero buffering capacity buffersuch as a carboxylic acid such as, but not limited to, formic acid,acetic, proprionic, butyric, citric, succinic, lactic or malic acidsacidified to a pH below about 4.0 by the addition of an inorganic acid;or at least one amino acid such as, but not limited to, glycine,alanine, valine, isoleucine, threonine or glutamine, methionine, serine,aspartic acid or at least one inorganic acid such as, but not limitedto, sulfuric acid, hydrochloric acid, nitric acid or phosphoric acid orany combination thereof. It is contemplated to be within the scope ofthe present invention for the acid moiety of the pharmaceutical carrierto be at least one physiologically tolerated buffer, oligopeptide,inorganic or organic ion or any combination thereof that will maintain apH in the pharmaceutically acceptable carrier below a value of about4.0.

[0050] The term “carbohydrate” as used herein refers to anypharmaceutically acceptable saccharide or disaccharide such as, but notlimited to, glucose, fructose, maltose, sucrose, lactose, trehalose,mannose, sugar alcohols including, but not limited to, sorbitol andmannitol, and polysaccharides such as, but not limited to, dextrins,dextrans, glycogen, starches and celluloses, or any combination orderivative thereof that are pharmaceutically acceptable to a human oranimal.

[0051] The term “stabilizing agent” as used herein refers to at leastone compound such as, but not limited to, polyhydric alcohols, glycerol,ascorbate, citrulline, niacinamide, glucosamine, thiamine, or inorganicsalt such as, but not limited to, sodium chloride, potassium chloride,calcium chloride, magnesium chloride or manganese chloride or anycombination thereof that will increase the stability of a preparation ofplasmin.

[0052] The term “reversibly inactivated acidified plasmin” as usedherein refers to any catalytically active form of plasmin capable ofproteolytically cleaving fibrin when under physiological conditions, butreversibly inactivated when placed at a pH between about pH 2.5 to about4.0. The term “inactivated” as used herein refers to a total orsubstantial reduction in enzymic activity compared to the activity atphysiological pH. The term “active plasmin” as used herein refers to aplasmin under conditions where the plasmin is capable of proteolyticallycleaving fibrin. The term “plasmin” includes, but is not limited toGlu-plasmin, Lys-plasmin, derivatives, modified or truncated variantsthereof. The term “truncated variants” includes, but is not limited to,midi-plasmin, mini-plasmin or the micro-plasmin as disclosed in U.S.Pat. No. 4,774,087 incorporated herein by reference in its entirety.

[0053] The term “anti-coagulant” as used herein refers to any compoundcapable of inhibiting the formation of a thrombus including, but notlimited to, hiruidin, heparin, thrombin inhibitors, platelet inhibitors,and any derivatives or combinations thereof.

[0054] The term “serine protease” as used herein refers to any serineprotease capable of proteoytically cleaving fibrin including, but notlimited to, plasmin, trypsin, chymotrypsin, pancreatic elastase II,cathepsin G, prostate-specific antigen, leukocyte elastase, chymase,tryptase, acrosin and human tissue kallikrein.

[0055] One limitation of current thrombolytic therapy with plasminogenactivators is plasminogen availability surrounding or within a thrombus.The local delivery of a fibrinolytic agent to a thrombus now allowsplasmin itself to be a potent therapeutic agent directly administered toa thrombus. In contrast to various plasminogen activators that arecurrently used as thrombolytics, direct localized thrombolytic therapywith plasmin can be intensified to whatever level is required to achieveclot lysis. This is because plasmin acts directly upon the fibrinpolymer. Also, plasmin, when delivered directly into or adjacent to athrombus, allows a lower effective dose to be administered with aconcomitant reduction in the systemic hemorrhage typically associatedwith conventional thrombolytic therapy. Excess plasmin can also berapidly inactivated by circulating α₂-antiplasmin.

[0056] The present invention contemplates that plasmin may be producedfrom plasminogen using any method that will yield a purified activeplasmin substantially free of plasminogen activator. It is within thescope of the present invention for the plasminogen to be any recombinantplasminogen or a truncated plasminogen such as, but not limited to, themini-plasminogen and micro-plasminogen, as disclosed by Wu et al. inU.S. Pat. No. 4,774,087 incorporated herein by reference in itsentirety. For example, Examples 1 and 2 below disclose a method wherebyactive plasmin was prepared from plasminogen purified from Cohn FractionII+III. The purity of the plasmin obtained using this method was greaterthan 95% and the specific activity was in the range of 18-23 CU/mg. Theplasmin preparations were substantially free of urokinase, or any otherplasminogen activator used for conversion of plasminogen into plasmin.

[0057] The plasmin of the present invention was purified by binding to abenzamidine affinity column and the subsequently eluted plasmin wascollected and stored in an acidified pharmaceutically acceptablecarrier. Results showed that a low pH in the range of about 2.5 to about4.0 greatly stabilized the plasmin composition, even when held at roomtemperature or greater. While not bound by any one theory, it isbelieved that at this low pH the plasmin has minimal serine proteaseactivity that would otherwise lead to autodegradation, as is seen whenplasmin is stored at physiological pH of between about 7.0 to about 7.5.

[0058] When the plasmin is administered directly into a thrombus, orproximal thereto, the plasmin encounters the physiological pH of theclot of about 7.4. The acidified pharmaceutically acceptable carrierattains the pH value of the thrombus, whereupon the plasmin recovers itsserine protease activity and begins to digest fibrin. Furthermore, thehigh concentration of fibrin within the thrombus provides an alternativesubstrate for the plasmin to minimize auto-degradation and maximizethrombolysis.

[0059] Fibrinolytic therapy employing a plasmin preparation that rendersplasmin proteolytically inactive until administered into, or immediatelyadjacent to, a thrombus and which is also substantially free of anyplasminogen activator reduces the likelihood of undesirable systemichemorrhage. Excess administered plasmin is rapidly inactivated bycirculating serum inhibitors such as α₂-antiplasmin, and the plasminogenactivators that would otherwise circulate to induce distal fibrinolysisare substantially absent.

[0060] Reversibly inactivated acidified plasmin, of the presentinvention may be readily stored, even at 37° C., in low bufferingcapacity pharmaceutically acceptable carriers such as, but not limitedto, 2 mM sodium acetate. Any pharmaceutically acceptable moiety may beused, singularly or in combination that maintains the composition at apH in the range of about 2.5 to about 4.0, especially at a pH of about3.1 to about pH 3.5. Acidic compounds useful in the present invention,either singly or any combination thereof, include but are not limited toformic acid, acetic acid, citric acid, hydrochloric acid, a carboxylicacid such as, but not limited to, lactic acid, malic acid, tartaricacid, benzoic acid, serine, threonine, methionine, glutamine, glycine,isoleucine, valine, alanine, aspartic acid, derivatives thereof, orcombinations thereof that will maintain the pH in the pharmaceuticallyacceptable carrier between about pH 2.5 to about pH 4.0.

[0061] The reversibly inactivated acidified plasmin composition of thepresent invention may further comprise at least one stabilizing agentsuch as a pharmaceutically acceptable carbohydrate including, but notlimited to, monosaccharides, disaccharides, polysaccharides, andpolyhydric alcohols. For example, pharmaceutically acceptablecarbohydrate stabilizers contemplated to be within the scope of thepresent invention include sugars such as, but not limited to, sucrose,glucose, fructose, lactose, trehalose, maltose and mannose, and sugaralcohols including, but not limited to, sorbitol and mannitol.Contemplated within the scope of the present invention arepolysaccharides such as, but not limited to, dextrins, dextrans,glycogen, starches and celluloses, or any combination thereofpharmaceutically acceptable to a human or animal patient.

[0062] Stabilizing agents contemplated as within the scope of thepresent invention and useful in stabilizing the reversibly inactivatedacidified plasmin composition of the present invention include, but arenot limited to, glycerol, niacinamide, glucosamine, thiamine, citrullineand inorganic salts such as, but not limited to, sodium chloride,potassium chloride, magnesium chloride, calcium chloride, or anycombination thereof. Other stabilizing agents contemplated as within thescope of the present invention may include, but are not limited to,pharmaceutically acceptable compounds such as benzyl alcohol or benzoicacid, to retard microbial contamination.

[0063] A plasmin composition according to the present invention can beadministered by any method that will deliver the plasmin as a bolus oras a prolonged infusion directly into a thrombus, or to a site a shortdistance proximal to the thrombus whereupon the plasmin composition canrapidly encounter the thrombus. By minimizing the distance from thecatheter to the thrombus, the reversibly inactivated acidified plasmincomposition's exposure to serum inhibitors is reduced. Catheter deliveryto a thrombus allows precision in placing the plasmin composition,especially within the thrombus.

[0064] A description of the method of treating a thrombotic occlusion ina patient using a therapeutically effective dose of the reversiblyinactivated acidified plasmin compositions of the present invention isdisclosed in U.S. patent application Ser. No. ______, entitled “Methodof Thrombolysis by Local Delivery of Reversibly Inactivated AcidifiedPlasmin”, commonly assigned and filed contemporaneously with the instantapplication, and is incorporated herein by reference in its entirety.

[0065] Additionally, a process for producing the reversibly inactivatedacidified plasmin composition of the instant invention is disclosed inU.S. patent application Ser. No. ______, entitled “Process for theProduction of a Reversible Inactivated Plasmin Composition”, commonlyassigned and filed contemporaneously with the instant application, andis incorporated herein by reference in its entirety.

[0066] Thus, the reversibly inactivated acidified plasmin composition ofthe present invention can be stored without a significant loss in theactivity of the plasmin restored by adjusting the pH of the compositionto physiological pH and safely used as a thrombolytic agent duringcatheter-assisted administration to a patient having a thromboticocclusion. The present invention is a plasmin composition substantiallyfree of plasminogen activators that exhibits at least comparablefibrinolytic activity to tPA and the safety profile appears at leastsimilar in this animal model of local thrombolytic delivery. It iscontemplated to be within the scope of the present invention that thereversibly inactivated acidified fibrinolytic enzyme may be, but is notlimited to, plasmin, derivatives of plasmin such as truncated formsthereof including, but not limited to, mini-plasmin and micro-plasmin asdisclosed by Wu et al. in U.S. Pat. No. 4,774,087 incorporated herein byreference in its entirety.

[0067] The present invention is further illustrated by the followingexamples, that are provided by way of illustration and should not beconstrued as limiting. Even though the invention has been described witha certain degree of particularity, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the present disclosure. Accordingly, it is intended thatall such alternatives, modifications, and variations that fall withinthe spirit and the scope of the invention be embraced by the definedclaims.

[0068] It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniquesdiscovered by the inventors to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. Those of skill in the art should, however, in light of thepresent disclosure, will appreciate that many changes can be made in thespecific embodiments disclosed and still obtain like or similar resultswithout departing, again, from the spirit and scope of the presentinvention. The contents of all references, published patents and patentscited throughout the present application are hereby incorporated byreference in their entirety.

EXAMPLES Example 1 Sources of Proteins Investigated

[0069] Plasminogen was purified from Cohn Fraction II+III paste byaffinity chromatography on Lys-Sepharose as described by Deutsch & Mertz(1970). Thus, 200 g of the paste was resuspended in 2 liter of 0.15Msodium citrate buffer, pH 7.8. The suspension was incubated overnight at37° C., centrifuged at 14,000 rpm, filtered through fiberglass and mixedwith 500 ml of Lys-Sepharose 4B (Pharmacia). Binding of plasminogen wasat room temperature for 2 hours. The Lys-Sepharose was then transferredonto a 2 liter glass filter, and washed several times with 0.15M sodiumcitrate containing 0.3M NaCl until the absorbance at 280 nm droppedbelow 0.05. Bound plasminogen was eluted with three 200 ml portions of0.2M ε-aminocaproic acid. Eluted plasminogen was precipitated with 0.4 gsolid ammonium sulfate/ml of plasminogen solution. The precipitate ofcrude (80-85% pure) plasminogen was stored at 4° C.

[0070] Low-molecular weight urokinase (LMW-urokinase) (Abbokinase-AbbottLaboratories, Chicago III.) was further purified by affinitychromatography on benzamidine-Sepharose. The Urokinase was then coupledto CNBr-activated Sepharose 4B by mixing 1.3 mg of LMW-urokinase in 50mM acetate buffer, pH 4.5, and diluting with 5 ml of the couplingbuffer, 0.1M sodium bicarbonate, pH 8.0.

[0071] This solution was immediately combined with 5 ml ofCNBr-activated Sepharose previously swollen and washed in 0.1M HCl. Thecoupling occurred for 4 hours on ice with shaking. The excess of theCNBr active group was blocked with 0.1M Tris, pH 8.0. Each batch ofurokinase-Sepharose was used 5 times and stored in 50% glycerol in waterat 4° C. between the cycles. Tissue plasminogen activator (Activase) wasfrom Genentech. Plasminogen-free fibrinogen and α-thrombin (3793 U/ml)were from Enzyme Research, Inc. α₂-Antiplasmin was obtained from AthensResearch Technologies. Commercially available plasmin was fromHaemotologic Technologies, Inc. Chromogenic plasmin substrate S2251 wasfrom Chromogenix. ¹²⁵I-Labeled human fibrinogen (150-250 μCi/mg) wasfrom Amersham Pharmacia Biotech. SDS-polyacrylamide gel electrophoresiswas performed in the Pharmacia Phast System apparatus using pre-made8-25% gradient gels and SDS-buffer strips. The source of plasminogen isnot limited to purification from a plasma source. It is contemplatedthat plasminogen may also be obtained from a transgenic or recombinantsource.

[0072] Example 2

Purification of Active Plasmin

[0073] (i) Activation of plasminogen to plasmin usingurokinase-Sepharose.

[0074] Plasminogen was cleaved to plasmin yielding plasmin withoutcontamination of the final preparation by using an immobilizedplasminogen activator. Urokinase cleaves plasminogen directly.Plasminogen activation by urokinase does not depend on the presence offibrin as in the case of tPA, and urokinase is a human protein. Thesefactors, and its relative low cost, make urokinase the preferredactivator, although this does not preclude the use of tPA, streptokinaseor any other cleavage means yielding an active plasmin capable of fibrindegradation. The ammonium sulfate precipitate of crude plasminogen wascentrifuged at 14,000 rpm and resuspended in a minimal volume using 40mM Tris, containing 10 mM lysine, 80 mM NaCl at pH 9.0 to achieve thefinal protein concentration of 10-15 mg/ml. The plasminogen solution wasdialyzed overnight against the same buffer to remove ammonium sulfate.The dialyzed plasminogen solution (10-20 ml) was diluted with an equalvolume of 100% glycerol and combined with 5 ml of urokinase-Sepharose.The use of 50% glycerol reduces autodegradation of plasmin duringactivation. Plasmin is stable in 50% glycerol and can be stored in thissolution at −20° C. for an extended period.

[0075] The plasminogen activation occurred at room temperature forbetween 2 hours and 24 hours depending on the freshness of theurokinase-Sepharose. With a fresh batch of urokinase-Sepharose,activation could be completed in 2 hours. It deteriorates, however, andbecomes less efficient after several cycles, necessitating the use ofSDS-PAGE under reducing conditions to monitor the progress ofplasminogen activation. Upon completion of the activation, the plasminsolution was filtered from the urokinase-Sepharose with a glass filter,and immediately applied to benzamidine-Sepharose.

[0076] (ii) Capturing of plasmin on benzamidine-Sepharose.

[0077] Since the plasmin is a serine protease with trypsin-likespecificity, benzamidine-Sepharose is an affinity absorbent that allowedcapture of the active plasmin. A plasminogen solution in 50% glycerolwas applied to the 50 ml benzamidine-Sepharose column equilibrated with0.05M Tris, pH 8.0, containing 0.5M NaCl with a flow rate of 3 ml/min.The column was run at 3 ml/min at 3-7° C. The front portion of thenon-bound peak contained high-molecular weight impurities. The rest ofthe non-bound peak is represented by residual non-activated plasminogenand by inactive autodegradation products of plasmin.

[0078] (iii) Elution of the bound plasmin with low pH buffer.

[0079] To protect plasmin from inactivation at neutral pH conditions,acidic elution conditions were selected. The plasmin bound tobenzamidine-Sepharose was eluted with 0.2M glycine buffer, pH 3.0containing 0.5M NaCl. The bound peak was typically divided into threepools, two front peaks, B1 and B2, and the bulk of the eluted materialas B3.

[0080] Non-reducing gel analysis showed that all three pools containedhighly pure (>95%) plasmin. The gel analysis, however, in addition tothe heavy and light chains of plasmin, revealed some low molecularweight bands in a range of 10-15 kDa as a result of partial internalcleavage degradation of the plasmin.

[0081] The front portion of peak B1 typically contained most of the lowmolecular weight impurities. The B2 and B3 pools were less degraded. Thefront portion of the bound peak had very little of the plasmin activityand was usually discarded. The loss of activity in this material may bedue to autodegradation during chromatography, because there is noglycerol present in the eluted material, and the pH of the front portionis intermediate between the pH of the equilibrating and eluting buffers,typically in a range of pH 6-6.5. The eluted plasmin, substantially freeof plasminogen activators, was collected in tubes containing 2M glycinebuffer, pH 3.0 (10% of the collected volume).

[0082] (iv) Formulation of eluted material in acidified water (pH 3.7).

[0083] Eluted plasmin was dialyzed with water and acidified to about pH3.7 with glacial acetic acid. Any acid providing a pharmaceuticallyacceptable acidified carrier having a low buffering capacity buffer andhaving a pH between about 2.5 to about 4.0 can be used. For example,also contemplated within the scope of this invention is the use of otheracids and amino acids such as, but not limited to, inorganic acids,carboxylic acids, aliphatic acids and amino acids including, but notlimited to, formic acid, acetic acid, citric acid, lactic acid, malicacid, tartaric acid, benzoic acid, serine, threonine, valine, glycine,glutamine, isoleucine, β-alanine and derivatives thereof, either singlyor any combination thereof, that will maintain the pH in thepharmaceutically acceptable carrier of about 2.5 to about 4.0.

[0084] Plasmin-specific activity was measured using an adaptedcaseinolytic assay as described by Robbins & Summaria (1970). One ml of4% casein solution in acidified water and an appropriate volume of 67 mMsodium phosphate buffer, pH 7.4 was added to a test polycarbonate tube.The solutions were vortexed and incubated at 37° C. for 10 minutes.Plasmin samples or buffer (blank) were added to each tube at 15 secondintervals, mixed thoroughly and incubated at 37° C. for 30 minutes. Thereaction was stopped with the addition of 3 ml of 15% trichloroaceticacid and the precipitate was allowed to form for 15 minutes. The tubeswere centrifuged at 3200 rpm for 20 minutes. The supernatants weretransferred to cuvettes and the A₂₈₀ of each sample was determined. Thespecific caseinolytic activity of each sample was determined by thefollowing formula:$\frac{3.27 \times \left\lbrack {{A_{280}\left( {{Plasmin}\quad {Sample}} \right)} - {A_{280}({Blank})}} \right\rbrack}{\mu \quad g\quad {Plasmin}\quad {in}\quad {Assay}} = {{CU}/{mg}}$

[0085] The plasmin concentration was determined spectrophotometricallyusing the extinction coefficient of 1.7 for 0.1% solution.

Example 3 pH-dependent Stability of Plasmin

[0086] Plasmin exhibits a bell-shaped pH dependence of its catalyticactivity. As shown in FIG. 1, plasmin has maximum enzyme activity at pH7.5-8.0, and its activity rapidly decreases at either more alkaline ormore acidic pHs. Plasmin is mostly inactive, and reversibly so, below pH4.0, due to the protonation of histidine in the catalytic center, asshown by Robbins & Summaria, (1976) and Castellino & Powell (1981).

[0087] Plasmin is very unstable at a physiological pH. Both the heavychain and light chains of plasmin degraded dramatically within hours atroom temperature and 4° C. Plasmin was formulated at 1 mg/ml in 0.04Msodium phosphate, pH 7.4, and incubated at 22° C. or 4° C. for 6 hours.During the incubation, the plasmin integrity was analyzed every twohours by reducing SDS-PAGE analysis. Both the heavy chain and lightchain degraded rapidly within hours at 22° C. and 4° C. as shown inTable 1. TABLE 1 The rapid degradation of plasmin in neutral pH solutionat 22° C. and 4° C. % of intact heavy chain % of intact light chainPlasmin Buffer PH Temp Initial 2 hr 4 hr 6 hr Initial 2 hr 4 hr 6 hr 1mg/ml 0.04M 7.4 22° C. 100% 27% 27% 29% 100% 29% 26% 28% PO₄ 1 mg/ml0.04M 7.4  4° C. 100% 32% 27% 25% 100% 33% 25% 22% PO₄

[0088] Plasmin at 1 mg/ml was incubated at 37° C. for 14 days underdifferent acidic conditions. The changes in plasmin heavy chain andlight chain were analyzed by running reducing SDS-PAGE. Plasmin wasformulated at 1 mg/ml in 0.04M sodium phosphate, pH 7.4 and was alsoincubated at 4° C. for six hours. During the incubation, the activity ofthe plasmin sample was measured every two hours by chromogenic potencyassay. Plasmin potency was quantitatively measured using the MLA 1600Canalyzer (Pleasantville, N.Y.). Plasmin hydrolyzed the chromogenicsubstrate S-2403 (D-pyroglutamyl-L-Phenylalanyl-L-Lysine-p-Nitroanilinehydrochloride or abbreviated as pyro-Glu-Phe-Lys-pNA) to form peptideand the chromophoric group p-nitroaniline (pNA). The rate of colorformation was measured kinetically at 405 nm. The amount of substratehydrolyzed was proportional to the plasmin activity in the sample. Astandard curve was generated from the linear regression of the rate ofcolor formation (OD/min) versus the potency of a plasmin standard. Thelinear equation together with the observed rate for an unknown samplewas used to calculate the potency of unknowns. The potency of plasminwas reported in units of mg/ml.

[0089] Plasmin integrity was significantly decreased by incubation at aphysiological pH, as shown in Table 2. TABLE 2 The rapid decrease ofplasmin activity in neutral pH solution at 4° C. Chromogenic PotencyPlasmin Buffer pH Initial 2 hr 4 hr 6 hr 1 mg/ml PO₄, 0.04M 7.4 100%43.3% 32.6% 26.4%

[0090] Plasmin formulated in acidified water at pH 3.7 is stable. It canbe kept in this form for months at reduced temperatures without any lossof activity or the appearance of degradation products of a proteolyticor acidic nature. FIG. 2 and the data of Table 3 show the stability ofplasmin at 4° C. and at room temperature. TABLE 3 Stability of 1 mg/mlplasmin in the following acidic conditions at 37° C. % intact heavy %intact light Plasmin chain after 14 days chain after 14 Formulation(mg/ml) Acidic Condition pH at 37° C. days at 37° C.  1 1 5 mM HAC/NaAc2.5 19% 62%  2 1 5 mM HAC/NaAc 3.0 41% 92%  3 1 5 mM HAC/NaAc 3.4 48%92%  4 1 5 mM HAC/NaAc 3.4 49% 96%  5 1 5 mM HAC/NaAc 3.4 50% 96%  6 1 5mM HAC/NaAc 3.7 13% 123%   7 1 5 mM HAC/NaAc 4.0 9.3%  107%   8 1 5 mMcitric  2.27 9.3%  64% acid/Na citrate  9 1 5 mM citric 3.1 33% 68%acid/Na citrate 10 1 5 mM citric  3.56 46% 88% acid/Na citrate 11 1 5 mMcitric 4.0 7.4%  104%  acid/Na citrate 12 1 5 mM glycine 2.2 7.3%  104% 13 1 5 mM glycine 3.1 36% 70% 14 1 5 mM glycine 3.5 49% 85% 15 1 5 mMglycine 3.8 12% 85% 16 1 5 mM glycine 4.1  6% 81% 17 1 5 mM serine 3.456% 100%  18 1 5 mM threonine 3.4 54% 100%  19 1 5 mM valine 3.4 52% 96%20 1 5 mM isoleucine 3.4 51% 100%  21 1 5 mM β-alanine 3.7 33% 90% 22 12 mM benzoic 3.5 42% 93% acid 23 1 2 mM lactic acid 3.5 45% 91% 24 1 2mM malic acid 3.5 50% 90% 25 1 2 mM tartaric acid 3.5 28% 87%

[0091] At 4° C., plasmin is stable for at least nine months. At roomtemperature, reversibly inactivated acidified plasmin is stable for atleast two months. To determine the optimal pH of different bufferingagents and the effect of buffer concentration on plasmin stability,compositions of 1 mg/ml of plasmin were prepared in 10 mM, 20 mM or 40mM sodium acetate or glycine at various pH values. The samples werestored at 37° C. for 7 days, and the relative amount of intact plasminheavy chain remaining was determined by densitometry of Coomassiestained SDS gels. Shown in FIG. 3 is a plot of pH versus percent heavychain relative to total protein in each lane of the SDS gels. Theresults demonstrate a pH stability optimum of about 3.1-3.5,irrespective of the type of buffer, or buffer concentration.

[0092] Long-term stability at room temperature is important because itwould make this formulation compatible with long regimens ofthrombolytic administration. For example, 36 hour administration ofthrombolytics such as tissue plasminogen activator or urokinase iscommon in treatment of peripheral arterial occlusions.

[0093] The ability of reversibly inactivated acidified plasmin to becomefully active upon transfer to physiological pH is evidenced by itsactivity in the caseinolytic assay and also in the ¹²⁵I-fibrin-labeledthrombolysis assays. Both of these assays are performed at pH 7.4, andthere was complete recovery of plasmin activity during the change of pHand passing through the isoelectric point (pH 5-5.5). The plasmin isformulated in a low buffering capacity solvent and, when added to abuffered solution such as plasma, it rapidly adopts the neutral orphysiological pH instantly and the precipitation that usuallyaccompanies the slow passage through the isoelectric point, does notoccur.

Example 4 Plasmin has the same Intrinsic Fibrinolytic Potency as aPlasminogen/plasminogen Activator Mixture

[0094] Plasmin has the same intrinsic fibrinolytic potency as aplasminogen/plasminogen activator mixture. Fibrinolytic potency ofplasmin was compared with that of a Lys-plasminogen and tPA mixture.These experiments were performed in a defined system consisting of an¹²⁵I-radiolabeled fibrin thrombus submersed in PBS. FIG. 4 shows that,in a buffered environment, thrombolysis achieved with plasmin is almostidentical to the Lys-plasminogen plus tPA mixture (curves a and b,respectively). At the same time, no thrombolysis was observed with tPAalone (curve c) or in the absence of any proteins (curve d). The dataobtained with tPA alone shows that its activity is dependent on itssubstrate, plasminogen, to be an effective thrombolytic.

[0095] These data indicate that, in the absence of inhibitors and otherprotein factors present in plasma, there is no difference in the abilityto lyse fibrin thrombi between purified plasmin and the combination oftPA and Lys-plasminogen. To assess the thrombolytic potency of activeplasmin, the ¹²⁵I-fibrin-labeled thrombolysis assay was performed withplasma thrombi in a plasma environment.

Example 5 Stabilization of Reversibly Inactivated Acidified PlasminComposition with a Sugar or Sugar Alcohol

[0096] Acidified plasmin compositions were formulated according to thepresent invention, as described n Examples 1 and 2, in 5 mM of aceticacid at pH 3.7 with 0.1M of maltose, mannitol, sucrose or sorbitol addedas a stabilizer. A plasmin composition formulated without any excipientwas included as a control. All samples were incubated at 37° C. for 7days and the change in plasmin integrity analyzed using SDS-PAGE underreducing conditions, as described in Example 2 above. FIG. 5demonstrates that the percent degradation of plasmin in the low pHcompositions formulated with a sugar or sugar alcohol are significantlyreduced, as compared to the control without a sugar or sugar alcohol.

Example 6 Stabilization of Reversibly Inactivated Acidified PlasminComposition with Non-carbohydrate Stabilizing Agents

[0097] Reversibly inactivated acidified compositions were formulated at1 mg/ml in 5 mM acetic acid, pH 3.7, according to the present invention,with 0.1M of glucosamine, niacinamide, citrulline or thiamine added as anon-carbohydrate stabilizer. A reversibly inactivated acidified plasminformulation without any excipient stabilizing agent was included as acontrol. All samples were incubated at 37° C. for 7 days and the changein plasmin integrity analyzed using SDS-PAGE under non-reducingconditions. Referring now to FIG. 6, all of the non-sugar stabilizingagents tested improved the stability of the reversibly inactivatedacidified plasmin composition at 37° C. over the 7 day test period.

[0098] A reversibly inactivated acidified plasmin compositions was alsoformulated at 1 mg/ml in 2 mM acetic acid, pH 3.4, according to thepresent invention, with 150 mM sodium chloride as a stabilizing agent.The same formulation, but without sodium chloride, was also prepared andincluded as a control. Samples were incubated at 4° C. for 28 days. Thechange in plasmin integrity was analyzed using SDS-PAGE undernon-reducing conditions, as described in Example 3 above. The activitywas assessed also as described in Example 3. Values were normalizedrelative to day 0 controls that were assigned a value of 100%. Theresults, as shown in Table 5, demonstrated that plasmin stored at 4° C.was more stable in the low-pH formulation containing sodium chloride.TABLE 5 Stability of reversibly inactivated acidified plasmincomposition (2 mM sodium acetate, pH 3.4) with or without 150 mM sodiumchloride, stored at 4° C. Sodium chloride % intact heavy % intact light% activity Concentration Plasmin chain after 28 chain after 28 after(mM) (mg/ml) days days 28 days 0 1 90 93 81 150 1 101 95 97

Example 7 Degradation Pattern of Reversibly Inactivated AcidifiedPlasmin Composition Characterized by N-terminal Sequencing

[0099] The degradation peptides of plasmin samples were characterized byN-terminal sequencing as follows. Plasmin compositions were formulatedat low pH values: a pH less than 2.5 and a pH of 3.5 and 3.8 containing2 mM acetic acid. The plasmin samples were analyzed using SDS-PAGE with4-12% Bis-Tris NuPage gels, as shown in FIG. 7. The protein bands weretransferred to a PVDF membrane, stained with Coomassie Blue R-250(Bio-RAD Laboratories, Hercules, Calif.) and bands cut out using ascalpel.

[0100] N-terminal sequence analysis was performed directly from themembrane using a Hewlett Packard 241 Protein Sequencer (Hewlett Packard,Inc., Glen Allen, Va.). Ten cycles were run for each band so that thecorresponding fragment of plasmin could be identified. Molecular weightsfor each band were determined with densitometry analysis using the Mark12 marker available from Invitrogen, Inc. (San Diego, Calif.)

[0101] Three polypeptides generated by incubation of plasmin at pH 3.8began at positions (numbering relative to Lys-plasmin) threonine (T105),glycine (G190) and glutamic acid (E623). From the known amino acidsequence of plasmin, it was determined that the first two polypeptideswere from the heavy chain and the third from the light chain. As shownin FIG. 8, the amino acid preceding the N-terminal amino acid was eitherarginine or lysine (K104, R189, and K622). It is commonly known thatplasmin cleaves proteins on the carboxyl side of lysine and arginine.These results demonstrated that compositions of plasmin at pH 3.8 weresusceptible to autodegradation.

[0102] Three polypeptides generated by incubation of plasmin at pH 2.2began with proline at the N-termini. From the known amino acid sequenceof plasmin, it was determined that these polypeptides were from theheavy chain, starting at positions P63, P155, and P347, as shown in FIG.8. The amino acid preceding each proline was an aspartic acid (D62,D154, and D346). It is commonly known that aspartyl-prolyl (D-P) peptidebonds are acid labile. These results demonstrated that compositions ofplasmin at pH 2.2 were susceptible to acid hydrolysis of peptide bonds.

Example 8 Low Buffer Capacity of Compositions

[0103] Plasmin (1 mg/ml) was formulated in 2 mM acetic, benzoic, lactic,malic or tartaric acid at pH 3.5. The effect of admixing increasingvolumes of human blood serum to the pH of 1 ml of the plasmin solutionwas measured (FIG. 9). In all cases, only a small amount of serum,typically 10 to 30% of the plasmin volume, was required to achieve a pHof about 7. In a separate experiment, plasmin compositions containedeither 5 or 10 mM acetic acid. The volumes of serum required toneutralize the plasmin solution were 30% and 70% of the initial volume.These results demonstrated that low buffering capacity compositions, oftypically around 2 mM, but upwards of 100 mM, are readily restored to apH of about 7. These results also suggest that plasmin would be readilyneutralized locally within a thrombus and that large volumes (relativeto the liquid fraction of the clot) of plasmin could be used to affectlysis.

Example 9 Lysis of Thrombi by Reversibly Inactivated Acidified Plasminin an in vitro Thrombus Model

[0104] To compare the efficacy of plasmin and tPA toward the lysis oflong retracted clots, we have developed an in vitro model which wouldmimic parameters of the clots formed in patients with PAO.

[0105] In Vitro PAO Model. Fresh whole human blood was collected into30×0.95 cm glass tubes and allowed to clot spontaneously withoutadditives. Tubes were incubated for 20 hours at 37° C. to allow fullretraction. Retracted clots were separated from serum using USA Standardtesting sieves D16 with 14 mesh and their weights were determined. Bloodclots were transferred into smaller diameter glass tubes that resembledthe average size clots in leg arteries (0.6×12 cm). A multi-side portpulse-spray catheter (French size 5 with the 11 cm spraying tip, Cook,Inc.) was inserted into the clot and a thrombolytic reversiblyinactivated acidified plasmin composition according to the presentinvention, or tPA) at 1 mg/ml was delivered in 1 ml increments separatedby 1 hour time intervals. The number of injections corresponds to thedose of thrombolytic. The extent of clot lysis was measured by theweight of a residual clot and expressed as a percent of clot weightreduction. Although this model is called a PAO model, and mimics thedimensions of the clots found in PAO patients, venous blood was used forclot formation. Both tPA and the reversibly inactivated acidifiedplasmin composition according to the present invention were tested inthis model and the results are presented below.

[0106] Plasmin is as effective as tPA for lysis of fresh clots, unlikewhen tPA and plasmin are used for lysis of retracted clots aged for 20hours to allow complete cross-linking by Factor XIII. tPA is unable tolyse such clots. Clot weight reduction obtained with tPA-treated clotsis similar to the control, even when the dose is raised to 5 mg perclot.

[0107] Plasmin, on the other hand, is effective toward both fullyretracted and cross-linked clots. There is a dose-dependence of thelytic effect of plasmin and after five injections (or 5 mg plasmin intotal) the clots are almost completely lysed. In a similar human seriesof experiments, the same inability to dissolve retracted andcross-linked clots was observed with urokinase. Locally deliveredplasmin therefore is a more effective thrombolytic agent than tPA andother plasminogen activators.

[0108] These in vitro data show that tPA requires the presence of itssubstrate, plasminogen, in the clot to initiate and maintain clot lysis.Therefore, while plasmin is as effective as tPA for lysing fresh orplasminogen-rich clots, plasmin is more effective that tPA, and otherplasminogen activators, for lysing of long retracted plasminogen-poorclots. Moreover, the data presented in this example demonstrates thatplasmin is effective in its reversibly inactivated acidified form whenit is injected directly inside the clot.

[0109] The PAO model as described above was used to compare the efficacyof plasmin (1 mg/ml) formulated in a low pH formulation described inthis invention (saline, pH 3.7) with a neutral pH plus stabilizerformulation. The results are demonstrated in FIG. 10 and show that thelow pH formulation is as efficacious as the neutral pH plus stabilizerformulation.

Example 10 Trypsin Stabilized at Low pH can be Reactivated by Transferto Higher pH Environment

[0110] Trypsin (16.4 mg, Sigma Chemical Co. Catalog No. T-1426) wasdissolved in 2.28 ml of 50 mM Tris/0.5M NaCl (pH 8.0). The trypsinsolution was loaded onto a 1-ml column of Benzamidine-Sepharose(Pharmacia Code No. 17-0568-01) that had been pre-equilibrated with 50mM Tris/0.5M NaCl (pH 8.0). This column was washed with 4 ml of thislatter buffer, resulting in a decrease in the eluate absorbance (280 nm)to less than 0.03. Trypsin was eluted from this column in an inactivatedstate with 0.5-ml volumes of 200 mM glycine/0.5M NaCl (pH 3.0); thethird through fifth 0.5-ml fractions eluted from this column containedthe peak values of absorbance (280 nm) and were pooled. The absorbance(280 nm) of this pooled trypsin eluate was determined to be 9.22; basedupon the extinction coefficient for trypsin (E₂₈₀ for a 1%solution=17.09) and the molecular weight of trypsin (24,000), theconcentration of total trypsin protein in this pooled column eluate wascalculated to be 225 μM.

[0111] The concentration of trypsin active sites in this pooled trypsincolumn eluate was determined by the method described by Case & Shaw,Biochem. Biophys. Res. Commun. 29, 508 (1967) and incorporated herein byreference in its entirety, using p-nitrophenylguanidinobenzoate asactive-site titrant. This assay was performed at pH 8.3, by diluting asmall volume (100 μl) of the pooled trypsin column eluate into an assaymixture also containing 700 μl of 50 mM sodium borate (pH 8.3), 200 μlof 10 mM sodium phosphate/1% glycine (pH 7.0) plus 10 μl ofp-nitrophenylguanidininobenzoate (dissolved in dimethyl formamide); thefinal pH of this mixture composition was determined to be 8.3. Thetrypsin-dependent amount of p-nitrophenol formed in this assay wasmonitored at 410 nm. Based upon the extinction coefficient forp-nitrophenol at 410 nm and at pH 8.3 (16,595M⁻¹), 100 μl of this pooledtrypsin column eluate present in the 1.01-ml assay corresponded to aconcentration of 22.95 μM trypsin active sites present in the cuvette.Therefore, the original stock solution of pooled trypsin column eluatecontained 231 μM trypsin active sites. This latter value is identical,within experimental error, to the concentration of total trypsin proteinpresent (225 μM). These results demonstrate that trypsin can be adjustedto low pH and then transferred to a higher pH environment withreactivation of its active site.

What is claimed is:
 1. A fibrinolytic composition comprising: areversibly inactive, acidified plasmin, the plasmin being substantiallyfree of a plasminogen activator; a low buffering capacity buffer; andoptionally, a stabilizing agent.
 2. The composition of claim 1, whereinthe plasmin is selected from Glu-plasmin, Lys-plasmin, midi-plasmin,mini-plasmin, or micro-plasmin.
 3. The composition of claim 1, whereinthe fibrinolytic composition is lyophilized.
 4. The composition of claim1, further comprising an aqueous carrier.
 5. The composition of claim 1,further comprising an anticoagulant.
 6. The composition of claim 1,wherein the fibrinolytic composition has a pH between about 2.5 andabout
 4. 7. The composition of claim 1, wherein the buffer comprises atleast one acid.
 8. The composition of claim 1, wherein the buffercomprises a carboxylic acid, at least one amino acid, a derivative ofthe at least one amino acid, a dipeptide, an oligopeptide which includesthe at least one amino acid, or a combination thereof.
 9. Thecomposition of claim 1, wherein the buffer is selected from formic acid,acetic acid, citric acid, hydrochloric acid, lactic acid, malic acid,tartaric acid, benzoic acid, serine, threonine, methionine, glutamine,alanine, glycine, isoleucine, valine, alanine, aspartic acid,derivatives thereof, or combinations thereof.
 10. The composition ofclaim 1, wherein the stabilizing agent is selected from a polyhydricalcohol, a salt, citrulline, or combinations thereof.
 11. Thecomposition of claim 1, wherein the plasmin is in the concentrationrange of between about 0.01 mg/ml to about 50 mg/ml.
 12. The compositionof claim 7, wherein the acid is in the concentration range of betweenabout 1 mM and about 100 mM.
 13. The composition of claim 1, wherein thestabilizing agent is a pharmaceutically acceptable carbohydrate, salt,glucosamine, thiamine, niacinamide, citrulline, or combinations thereof.14. The composition of claim 1, wherein the stabilizing agent is a sugaror sugar alcohol selected from glucose, maltose, mannitol, sorbitol,sucrose, lactose, trehalose, or combinations thereof.
 15. Thecomposition of claim 1, wherein the stabilizing agent is selected frommonosacchrides, disaccharides, polysaccharides, polyhydric alcohols, orcombinations thereof.
 16. The composition of claim 13, wherein thecarbohydrate has a concentration in the range of about 0.2% w/v to about20% w/v.
 17. The composition of claim 1, wherein the stabilizing agentis selected from sodium chloride, potassium chloride, magnesiumchloride, calcium chloride, manganese chloride or combinations thereof.18. The composition of claim 1, wherein the stabilizing agent isselected from a salt, glucosamine, thiamine, niacinamide, or acombination thereof and the concentration of the stabilizing agent is inthe range of about 0.01M to about 1M.
 19. The composition of claim 1,wherein the buffer is present in the composition at a concentration atwhich the pH of the composition is raised to a physiological pH bycontacting a body fluid or a thrombus.
 20. A fibrinolytic compositioncomprising: a reversibly inactive, acidified serine protease, the serineprotease being substantially free of a plasminogen activator; a lowbuffering capacity buffer; and optionally, a stabilizing agent.
 21. Thecomposition of claim 20, wherein the fibrinolytic composition islyoplilized.
 22. The composition of claim 20, further comprising anaqueous carrier.
 23. The composition of claim 20, further comprising ananticoagulant.
 24. The composition of claim 20, wherein the fibrinolyticcomposition has a pH between about 2.5 and about
 4. 25. The compositionof claim 20, wherein the buffer comprises at least one acid.
 26. Thecomposition of claim 20, wherein the low buffering capacity buffercomprises a carboxylic acid, at least one amino acid, a derivative ofthe at least one amino acid, a dipeptide, an oligopeptide which includesthe at least one amino acid, or a combination thereof.
 27. Thecomposition of claim 20, wherein the buffer is selected from aceticacid, citric acid, hydrochloric acid, lactic acid, malic acid, tartaricacid, benzoic acid, serine, threonine, methionine, glutamine, alanine,glycine, isoleucine, valine, alanine, aspartic acid, derivativesthereof, or combinations thereof.
 28. The composition of claim 20,wherein the stabilizing agent is selected from a polyhydric alcohol, asalt, citrulline, or combinations thereof.
 29. The composition of claim20, wherein the plasmin is in the concentration range of between about0.01 mg/ml to about 50 mg/ml.
 30. The composition of claim 25, whereinthe acid is in the concentration range of between about 1 mM and about100 mM.
 31. The composition of claim 20, wherein the stabilizing agentis a pharmaceutically acceptable carbohydrate, salt, glucosamine,thiamine, niacinamide, citrulline, or combinations thereof.
 32. Thecomposition of claim 20, wherein the stabilizing agent is a sugar orsugar alcohol selected from glucose, maltose, mannitol, sorbitol,sucrose, lactose, trehalose, or combinations thereof.
 33. Thecomposition of claim 20, wherein the stabilizing agent is selected frommonosacchrides, disaccharides, polysaccharides, polyhydric alcohols, orcombinations thereof.
 34. The composition of claim 31, wherein thecarbohydrate has a concentration in the range of about 0.2% w/v to about20% w/v.
 35. The composition of claim 20, wherein the stabilizing agentis selected from sodium chloride, potassium chloride, magnesiumchloride, calcium chloride, or combinations thereof.
 36. The compositionof claim 20, wherein the stabilizing agent is selected from a salt,glucosamine, thiamine, niacinamide, or a combination thereof and theconcentration of the stabilizing agent is in the range of about 0.01M toabout 1M.
 37. The composition of claim 20, wherein the buffer is presentin the composition at a concentration at which the pH of the compositionis raised to a neutral pH by adding no more than about an equal volumeof serum to the composition.
 38. The composition of claim 20, whereinthe serine protease is selected from trypsin, chymotrypsin, pancreaticelastase II, cathepsin G, prostate-specific antigen, leukocyte elastase,chymase, tryptase, acrosin, human tissue kallikrein, and plasmin. 39.The composition of claim 38, wherein the plasmin is a truncated variantthereof selected from midi-plasmin, mini-plasmin, or micro-plasmin.